features of ras activation by a mislocalized oncogenic tyrosine … · 2013. 10. 9. · journal of...
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Features of Ras activation by a mislocalized oncogenictyrosine kinase: FLT3 ITD signals through K-Ras at theplasma membrane of acute myeloid leukemia cells
Susanne Kothe1, Jorg P. Muller1, Sylvia-Annette Bohmer1, Todor Tschongov1, Melanie Fricke1, Sina Koch2,3,Christian Thiede2, Robert P. Requardt4, Ignacio Rubio1,4 and Frank D. Bohmer1,*1Institute of Molecular Cell Biology, Center for Molecular Biomedicine, Jena University Hospital, 07743 Jena, Germany2Molecular Hematology Group, Department of Internal Medicine I, University Hospital Carl Gustav Carus, 01307 Dresden, Germany3Department of Systemic Cell Biology, Max Planck Institute for Molecular Physiology, 44227 Dortmund, Germany4Center for Sepsis Control and Care (CSCC), Jena University Hospital, 07743 Jena, Germany
*Author for correspondence ([email protected])
Accepted 24 July 2013Journal of Cell Science 126, 4746–4755� 2013. Published by The Company of Biologists Ltddoi: 10.1242/jcs.131789
SummaryFMS-like tyrosine kinase 3 with internal tandem duplication (FLT3 ITD) is an important oncoprotein in acute myeloid leukemia (AML).Owing to its constitutive kinase activity FLT3 ITD partially accumulates at endomembranes, a feature shared with other disease-associated, mutated receptor tyrosine kinases. Because Ras proteins also transit through endomembranes we have investigated the
possible existence of an intracellular FLT3-ITD/Ras signaling pathway by comparing Ras signaling of FLT3 ITD with that of wild-typeFLT3. Ligand stimulation activated both K- and N-Ras in cells expressing wild-type FLT3. Live-cell Ras–GTP imaging revealed ligand-induced Ras activation at the plasma membrane (PM). FLT3-ITD-dependent constitutive activation of K-Ras and N-Ras was also
observed primarily at the PM, supporting the view that the PM-resident pool of FLT3 ITD engaged the Ras/Erk pathway in AML cells.Accordingly, specific interference with FLT3-ITD/Ras signaling at the PM using PM-restricted dominant negative K-RasS17N potentlyinhibited cell proliferation and promoted apoptosis. In conclusion, Ras signaling is crucial for FLT3-ITD-dependent cell transformationand FLT3 ITD addresses PM-bound Ras despite its pronounced mislocalization to endomembranes.
Key words: Oncogenic RTK, FLT3, FLT3 ITD, Leukemia, AML, Ras, Localization, Plasma membrane, Endomembrane
IntroductionConstitutively active receptor tyrosine kinases (RTKs) play a
pathogenic role in a number of human diseases, including several
malignancies. They are active in a ligand-independent manner,
leading to aberrant and constitutive activation of downstream
signaling pathways. RTKs are N-glycosylated proteins that
undergo quality control in the endoplasmic reticulum (ER) and
maturation of their carbohydrate moiety while trafficking in
vesicles from ER through the Golgi to the plasma membrane
(PM) (Helenius and Aebi, 2004). For a number of constitutively
active RTKs such as constitutively active versions of FMS-like
tyrosine kinase 3 (FLT3), c-Kit/Stem-cell factor receptor,
anaplastic lymphoma kinase (ALK), fibroblast growth factor
receptor 3 (FGFR3), and the angiopoietin receptor tyrosine
kinase with immunoglobulin and epidermal growth factor
homology domains-2 (TIE-2) the normal processing along this
pathway is impaired (Choudhary et al., 2009; Lievens et al.,
2006; Limaye et al., 2009; Mazot et al., 2011; Schmidt-Arras
et al., 2005; Tabone-Eglinger et al., 2008; Xiang et al., 2007).
Inefficient processing is caused by yet unknown mechanisms, but
appears to be causally associated with constitutive tyrosine
kinase activity. As a consequence, these RTKs are partially
mislocalized in that they accumulate in intracellular ER/Golgi
compartments. Importantly, both the intracellular and the PM
pools of these RTKs can activate signal transduction. Differences
in signaling quality of both pools have been identified, notably a
preferred activation of STAT5 by intracellular RTK species
(Choudhary et al., 2009; Schmidt-Arras et al., 2009; Schmidt-
Arras et al., 2005).
FLT3 with internal tandem duplication of amino acid stretches
in the juxtamembrane or kinase domain (FLT3 ITD), is a
constitutively active version of FLT3 occurring in as many as
25–30% of patients with acute myeloid leukemia (AML)
(Breitenbuecher et al., 2009; Frohling et al., 2005; Kiyoi et al.,
1998; Meshinchi and Appelbaum, 2009; Thiede et al., 2002).
AML is the most frequent form of acute leukemias in adults and
arises by malignant transformation of myeloid progenitor cells
(Dohner et al., 2010). Mutations in FLT3 confer a proliferative
advantage and are referred to as ‘class I mutations’. In addition to
class I mutations, leukemogenesis requires further genetic lesions
such as mutations that lead to blocked hematopoietic
differentiation (‘class II mutations’) (Deguchi and Gilliland,
2002; Ishikawa et al., 2009; Cancer Genome Atlas Research
Network, 2013). Another group of class I mutations are activating
mutations in RAS genes (Frohling et al., 2005; Tyner et al., 2009).
RAS and FLT3-activating mutations are mutually exclusive,
indicating functional equivalence in the pathogenesis of leukemia
(Cancer Genome Atlas Research Network, 2013; Schlenk et al.,
2008). Consistent with this notion, previous studies have
suggested that Ras activation downstream of FLT3 ITD is
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essential for cell transformation, in addition to activation ofSTAT5 and of the AKT/phosphoinositide 3-kinase (PI3K)
pathway (Mizuki et al., 2000; Muller-Tidow et al., 2002;Takahashi, 2006; Toffalini and Demoulin, 2010). However,activation of Ras downstream of FLT3 ITD has never been
directly analyzed, either biochemically or in terms of itsspatiotemporal features. In particular it is not known whetherFLT3 ITD addresses Ras at endomembranes or at the PM.
The three prototypical Ras proteins H-Ras, K-Ras and N-Ras(collectively referred to as Ras, from here on) are differentiallyexpressed in different types of cells and tissues. Leukemia cells
express predominantly N- and K-Ras with little or no expressionof H-Ras (Omerovic et al., 2008). Furthermore, the various Rasisoforms exhibit distinct subcellular localization and traffickingproperties owing to differences in the pattern of post-translational
modifications (Omerovic et al., 2008). Notably, K-Ras islocalized almost entirely to the PM, whereas N-Ras and H-Rasshuttle back and forth between the PM and endomembranes in
the context of a palmitoylation/depalmitoylation cycle (Rockset al., 2010). Activation of all three isoforms of Ras proteins canbe assessed biochemically by employing the Ras-binding domain
(RBD) of the Ras effector Raf as an affinity probe for Ras–GTPisolation. This strategy can also be exploited for visualizing thesubcellular sites of Ras–GTP accumulation in intact cells byusing fluorescently labeled RBD reporter probes (Augsten et al.,
2006; Chiu et al., 2002; Rubio et al., 2010). In accordance withthe notion that de-palmitoylated versions of H-Ras and N-Rastransit through Golgi and ER (collectively known as
endomembranes) on their way to the PM (Choy et al., 1999),several studies reported the presence of active GTP-loaded Ras atendomembranes of growth factor challenged cells (reviewed by
Fehrenbacher et al., 2009). However, although there is agreementthat activation of Ras downstream of multiple types of receptorsoccurs initially at the PM, there is an ongoing controversy
regarding the possible existence of meaningful amounts of Ras–GTP at intracellular sites, a debate fueled by the fact thatendomembrane Ras activation has been detectable in cells thatoverexpress Ras (Chiu et al., 2002) but could not be observed in
the case of native, endogenous Ras (Augsten et al., 2006;Fehrenbacher et al., 2009; Rubio et al., 2010).
Given the importance of Ras for FLT3-mediated transformation,we have systematically analyzed Ras signaling downstream ofwild-type FLT3 and FLT3 ITD. Notably, we considered thepossibility that FLT3 ITD, which is partially retained in
intracellular membranes, could potentially functionally interactwith endomembrane-resident Ras. Using a combination ofbiochemical and fluorescence live-cell microscopy approaches,
we assessed the role of the different Ras isoforms in wild-typeFLT3 and FLT3 ITD signaling. The results revealed that Rasactivation takes place at the PM both for wild-type FLT3 and FLT3
ITD, and that K-Ras is the predominant Ras isoform activateddownstream of the transforming variant FLT3 ITD. Moreover, Rasactivation at the plasma membrane is required for cell proliferation
and viability.
ResultsDifferent localization of wild-type FLT3 and FLT3 ITD inhuman AML cell lines
To assess Ras activation downstream of FLT3, we chose twohuman leukemia cell lines, which harbor the same chromosomaltranslocation (4;11) leading to expression of a leukemia-associated
fusion protein (MLL-AF4), but differ in their FLT3 status. RS4-11
cells express wild-type FLT3, whereas MV4-11 cells express
solely FLT3 ITD. As shown in Fig. 1, localization of FLT3 in
these cell lines is clearly different. Immunostaining and
microscopy using confocal laser scanning microscopy (LSM)
revealed that FLT3 in RS4-11 is predominantly localized at the
PM, while FLT3 ITD in MV4-11 cells is to a large extent localized
in intracellular membranes (Fig. 1A). Quantitative analyses using
antibodies detecting an epitope in the FLT3 extracellular domain
(CD135 antibody) and flow cytometry were in excellent agreement
with this observation. A large fraction of FLT3 was detectable at
the surface of RS4-11 cells and much less FLT3 ITD at the surface
of MV4-11 cells. However, in both cell lines similar amounts of
FLT3 became detectable upon permeabilization of the cells
(Fig. 1B). We also employed 32D cells, an IL-3-dependent
murine myeloid cell line, stably transfected with either wild-type
FLT3 or FLT3 ITD for some analyses. A different localization of
wild-type FLT3 and FLT3 ITD in these cell lines has been reported
previously (Schmidt-Arras et al., 2009), with FLT3 ITD expressed
at endomembranes and to a lesser extent at the cell surface.
K-Ras and N-Ras are activated downstream of wild-type
FLT3
To evaluate which Ras isoforms are activated downstream of
the native, wild-type FLT3 receptor we performed biochemical
experiments using both 32D cells stably transfected with
Fig. 1. Aberrant localization of FLT3 ITD in intracellular membranes.
(A) The human leukemia cell lines RS4-11 and MV4-11 harboring
endogenous wild-type FLT3 or FLT3 ITD, respectively, were subjected to
immunostaining with anti-FLT3 antibodies (red). Plasma membranes were
decorated with Alexa-Fluor-488-labeled wheat germ agglutinin (WGA,
green). Nuclei were stained with HOECHST 33347 (blue). Scale bars: 5 mm.
(B) Quantitative analysis of FLT3 distribution, by flow cytometry. FLT3 was
detected using antibodies (anti-CD135) recognizing a surface epitope. Cells
were stained either intact, or after permeabilization to reveal the total FLT3
amounts, as indicated. The relative amount of surface FLT3 in the two cell
lines is also given.
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wild-type FLT3, and the human cell line RS4-11. As a first step, weanalyzed the expression profile of H-Ras, K-Ras and N-Ras in
these cell types. Although K-Ras and N-Ras proteins were presentat similar levels in both myeloid cell lines (although detected
expression was somewhat variable in different experiments,data not shown), H-Ras expression was not detectable byimmunoblotting (data not shown), a pattern found also in other
leukemia lines (Omerovic et al., 2008). Therefore, we confined ouranalysis to K-Ras and N-Ras. Activation of K-Ras and N-Ras was
analyzed by using a glutathione S-transferase (GST)–RBD affinityprobe to pull out active Ras from cell extracts (see Materials and
Methods). The presence of activated K- or N-Ras proteins wassubsequently detected by immunoblotting with isoform-specificRas antibodies. Cells were starved of serum (and of IL3 in the case
of 32D cells), and stimulated with FLT3 ligand (FL) for differentperiods of time. As shown in Fig. 2 (FLT3 IP panels), this led to a
rapid activation of FLT3, which remained high for up to10 minutes of stimulation and was still detectable 30 minutesafter ligand addition in RS4-11 cells. Both Ras isoforms, K-Ras
and N-Ras, were activated downstream of FLT3 with similarkinetics to that of FLT3 activation (Fig. 2, Ras-GTP pulldown
panels). Stimulation was also associated with a rapid activation ofErk1/2 (Fig. 2, lysate panels), which declined after 30 minutes.
Thus, these experiments showed that ligand-stimulated wild-typeFLT3 activates K-Ras and N-Ras with similar efficiency in twodifferent cell models.
Whereas K-Ras activation is largely restricted to the PM in allexperimental systems tested so far, N-Ras–GTP accumulation
has been documented both at the PM and at endomembranes ofstimulated cells (Chiu et al., 2002; Perez de Castro et al., 2004).To assess the localization of FLT3-induced K-Ras and N-Ras
activation, we monitored the subcellular distribution of E3-R3(A/D), a trivalent fluorescent affinity probe for Ras–GTP (see the
Materials and Methods for more details; shown in the figures ingreen; RBD) that has previously been employed to image Ras
activation in a number of settings (Augsten et al., 2006;Leadsham et al., 2009; Rubio et al., 2010; Wu et al., 2010).The specificity of this probe for detection of activated Ras
proteins in the employed cell system was confirmed by co-transfection with different N-Ras versions (supplementary
material Fig. S1). To analyze FLT3-dependent Ras activation,RS4-11 cells were co-transfected with green fluorescent E3-R3(A/D) and red fluorescent Ras proteins (mCherry Ras, labeled
in the Figures red as K- or N-Ras), challenged with FL andimaged confocally. As shown in Fig. 3, E3-R3(A/D) probe
redistribution can be seen upon FL stimulation over time. Intenselabeling of surface membranes by the E3-R3(A/D) probe was
apparent both in the case of mCherry–K-Ras-expressing(Fig. 3A) and mCherry–N-Ras-expressing (Fig. 3B) cells. Forboth isoforms, Ras activation at the PM was abundant and
relatively rapid. In the case of N-Ras an accumulation of E3-R3(A/D) at intracellular compartments was also visible (Fig. 3B).
However, a quantitative analysis of cells expressing mCherry–N-Ras revealed that ,50% of cells featured those intracellular
E3-R3(A/D)-illuminated dense and compact structures alreadybefore FL stimulation, whereas in 30–40% of cells E3-R3(A/D)decorated intracellular structures after FL stimulation. In these
cells, accumulation of E3-R3(A/D) at endomembranes was mostprominent and continued to rise at 60 minutes post-stimulation, a
time point at which little Ras-GTP is left in the cell as judged bythe biochemical analysis (Fig. 2). Thus, we assume that the
illumination of intracellular structures by E3-R3(A/D) may have
been provoked by the overexpression of mCherry–N-Ras. To
further characterize the intracellular structures that harbor the
RBD probe, their potential colocalization with several cellular
compartments was assessed. We detected some overlap in
localization of the compact probe accumulations with
endosomal vesicles (supplementary material Fig. S2). In sum,
these experiments confirmed that ligand-stimulated FLT3
activates both Ras isoforms, K-Ras and N-Ras, and that ligand-
dependent Ras activation proceeded predominantly at the PM.
K-Ras is prominently activated downstream of FLT3 ITD
We were particularly interested in Ras activation downstream of
FLT3 ITD since this pathway is considered to be critical for cell
transformation. For the biochemical assays, we employed the
murine cell line 32D, stably transfected with FLT3 ITD, and the
human cell line MV4-11, which endogenously expresses FLT3
ITD. Constitutive FLT3 phosphorylation was abundant in both
cell lines (Fig. 4A,B, FLT3 IP panels). A moderate constitutive
Fig. 2. FL stimulation of wild-type FLT3-expressing myeloid cells causes
K-Ras and N-Ras activation. (A,B) Wild-type murine FLT3 (mFLT3)-
expressing 32D cells (A) and human RS4-11 cells (B) endogenously
expressing wild-type FLT3 were treated with FL (20 ng/ml) or were left
untreated. At the indicated time points, cells were lysed and subjected to Ras–
GTP pulldown (PD). The activation status of Ras, FLT3 and ERK was
sequentially determined in the same lysates, as described in Materials and
Methods. The same membrane sections were reprobed to detect the different
Ras isoforms and to detect total proteins with pan-specific antibodies.
Antibodies used for immunoprecipitation (IP) or immunoblotting (left side of
panels) are indicated. Four (A) or two (B) independent experiments,
respectively, yielded consistent results. pErk1/2, anti-phospho-p44/42 MAPK;
pTyr591, anti-phospho-FLT3Y591. Note that FLT3 gives rise to two species,
a 130 kDa immature form, and a 150 kDa complex glycosylated form
(Schmidt-Arras et al., 2005). Only the latter is available at the
cell surface for the ligand and autophosphorylates in response to ligand
stimulation. Owing to high expression levels of mFLT3 in the 32D cell line
employed for the biochemical experiments (Grundler et al., 2005), relatively
large amounts of immature 130 kDa FLT3 are detectable in these cells, also
showing some basal autophosphorylation.
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Ras activation was also detectable for both isoforms and both cell
lines (Fig. 4A,B, Ras-GTP pulldown panels). This is consistent
with earlier reports on constitutive Erk1/2 activation in FLT3-
ITD-expressing cells (Mizuki et al., 2000), which we also
observed in both analyzed cell lines (Fig. 4A,B, lower panels).
To test the dependence of Ras activation on upstream activity of
FLT3 ITD, we treated the cells with the FLT3-selective tyrosine
kinase inhibitor cpd.102 (Mahboobi et al., 2006). Then, the
compound was washed out and Ras and Erk activation were
monitored over time. Indeed, kinase inhibition led to complete
inhibition of FLT3 autophosphorylation in both cell lines
as shown by immunoblotting. Identical results were obtained
by probing FLT3 phosphorylation with the site-selective
antibody pTyr591 (Fig. 4A,B) or an antibody against total
phosphotyrosine (pY100, data not shown). In the presence of
inhibitor, basal Ras activation was reduced in the case of K-Ras,and affected to a lesser extent in the case of N-Ras (Fig. 4C).Interestingly, Ras activation was strongly elevated after removal
of the inhibitor. In contrast to the basal state, under theseconditions, the magnitude of Ras activation was similar for bothisoforms. At the level of Erk1/2 phosphorylation, a pronouncedactivation could also be observed after inhibitor washout
(Fig. 4A,B, lysate panels), consistent with the pattern of Rasactivation.
Both cell lines were also subjected to the microscopic analysis
of spatial aspects of Ras activation. Prominent K-Ras activationat the PM was readily observed in both cell lines and wascompletely suppressed by treating the cells with the FLT3 kinaseinhibitor cpd.102 (Fig. 5A–E). To substantiate that K-Ras
activation occurs by the PM-bound pool of FLT3 ITD, weemployed a 32D cell line harboring a FLT3 ITD version that iscompletely trapped in endomembranes owing to the inclusion of
an ER-retention signal (32D FLT3 ITD R3) (Schmidt-Arras et al.,2009). Indeed, no K-Ras activation was detectable in these cells(Fig. 5C).
Assessment of N-Ras activation by FLT3 ITD at the PM
revealed that N-Ras activation was clearly weaker than activationof K-Ras, despite similar amounts of N-Ras or K-Ras (detectableas mCherry staining) in this location. In contrast, intracellular
dense and compact structures (‘spots’) of the E3-R3(A/D) probedetecting activated N-Ras were frequently observed (Fig. 5F–I),raising the possibility that intracellular located N-Ras was being
activated by FLT3 ITD. A quantitative comparison of theobserved activation of K-Ras and N-Ras in the different cellularlocalizations is shown in Fig. 5J,K. To test the hypothesis that the
apparent intracellular N-Ras activation in compact structures wasdriven by FLT3 ITD signaling, we again employed the FLT3inhibitor. Treatment with cpd.102, however, did not significantlyaffect the colocalization of E3-R3(A/D) and mCherry–N-Ras at
intracellular spots (Fig. 5F,H, and quantification in Fig. 5G,I).Moreover, in parental 32D cells co-expressing mCherry–N-Rasand E3-R3(A/D), formation of similar spot-like structures with
apparently activated N-Ras could be observed (not shown).Notably, as these cells do not express FLT3, N-Ras activationoccurred independently of FLT3 activation. Taken together our
results show that constitutively active FLT3 ITD clearly activatedRas at the PM, a phenomenon that was best seen for K-Ras. Someapparent intracellular accumulation of N-Ras–GTP could not,
however, be causally linked to FLT3 ITD activity. Obviously,despite an intracellular mislocalization of a large fraction ofFLT3 ITD, the oncoprotein appears incapable of activating N-Ras in endomembranes.
Activation of the plasma membrane pool of Ras is crucialfor FLT3-ITD-mediated cell growth and cell survival
K- and N-Ras may play different roles downstream of FLT3 ITD.
Also, PM and endomembrane pools of N-Ras could each transmitdistinct signals in the context of FLT3-ITD-dependenttransformation. In order to analyze the functional importance of
the specific Ras isoforms for FLT3-ITD-mediated leukemic celltransformation, we initially employed a siRNA strategy. Toselectively deplete K- or N-Ras or both Ras isoforms together
in FLT3-ITD-expressing MV4-11 cells, Ras-isoform selectivesiRNAs were used. For comparison, cells were also depleted ofFLT3 ITD by siRNA, or treated with the FLT3 kinase inhibitor
Fig. 3. FL stimulation of wild-type FLT3-expressing myeloid cells
promotes K-Ras and N-Ras activation at the plasma membrane. RS4-11
cells expressing endogenous wild-type FLT3 were co-transfected with
constructs encoding mCherry–K-Ras (A) or mCherry–N-Ras (B) (both shown
in red), along with E3-R3(A/D) (green, RBD), a trivalent fluorescent reporter
probe for Ras–GTP. Cells were deprived of serum for 2 hours, challenged
with FL (100 ng/ml), and analyzed by confocal laser-scanning microscopy.
Of at least 20 monitored cells each, ,40% (A) and ,70% (B) showed FL-
dependent redistribution of E3-R3(A/D) to the plasma membrane, indicating
Ras activation at that site, as shown in these examples. Scale bars: 10 mm.
Original magnification 636. In A, a larger area is shown to depict several
cells with similar reactions. Note that some of the probe accumulates in the
nucleus. The reason for this is not known, but it is unrelated to Ras activation
(Augsten et al., 2006).
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cpd.102 or the MEK inhibitor UO126. As biological end points
cell proliferation/viability and cell cycle progression were
analyzed. As shown in supplementary material Fig. S3,
although selective and efficient knockdown of the two Ras
isoforms K-Ras and N-Ras was achieved, the effects of the Ras
knockdowns on proliferation/viability and cell cycle progression
were only very mild. In contrast, siRNA-mediated knockdown
of FLT3 ITD or inhibition of FLT3 ITD kinase or MEK kinase
by pharmacological inhibitors very strongly impaired cell
proliferation/viability (supplementary material Fig. S3). We
hypothesized that the low levels of Ras isoforms remaining in
the siRNA-treated cells were presumably sufficient to still sustain
FLT3-ITD-dependent cell proliferation and survival. Therefore,
to rigorously prove that Ras played an important role in FLT3
signaling we resorted to a previously successfully applied
dominant negative (dn) Ras strategy. However, using the
commonly employed dn H-RasS17N (Mizuki et al., 2000;
Muller-Tidow et al., 2002) to interfere with Ras signaling, it is
not possible to discriminate the roles of differently localized Ras
pools, because dn H-RasS17N blocks signal transduction through
all three Ras isoforms and in all their cellular locations
(Matallanas et al., 2003). Therefore, we used dn K-RasS17N.
This molecule (like its wild-type counterpart) exclusively
localizes to plasma membranes (Omerovic et al., 2008) and
thus can interfere with Ras–GTP loading only in this location.
MV4-11 cells were infected with lentiviral particles encoding
EYFP-tagged K-Ras, the dn EYFP-tagged K-RasS17N version,
or the yellow fluorescent protein Venus as a control.
Subsequently, DNA synthesis was assessed using the Click-iT
labeling technology and flow cytometric analysis of the
transduced cell pool. Transduction with the dn K-RasS17N
construct greatly reduced the number of proliferating viable cells,
whereas wild-type K-Ras expression had no effect (Fig. 6A). In
addition to impairment of cell proliferation, dn K-Ras expression
caused extensive apoptosis, as assessed by annexin-V staining
(Fig. 6B,C). Taken together, the interference with Ras signaling
at the PM by expression of dn K-Ras abolished FLT3-ITD-
dependent cell growth and survival.
DiscussionThis study undertook the systematic comparison of biochemical
and spatiotemporal features of Ras activation downstream of
the hematopoietic RTK FLT3 and its leukemia-associated
counterpart FLT3 ITD. Main findings include the reduced
capacity of FLT3 ITD to activate N-Ras compared with wild-
type FLT3, and the observation that FLT3 ITD-mediated
Ras activation at the plasma membrane is predominant and
required for cell transformation. Importantly, the mislocalized
intracellular pool of activated FLT3 ITD appears incapable of
activating Ras in endomembranes.
Fig. 4. Constitutive K-Ras activation in FLT3-ITD-expressing myeloid cells depends on FLT3 kinase activity. Murine FLT3-ITD-expressing 32D cells
(A) or human MV4-11 cells endogenously expressing FLT3 ITD (B) were serum starved for 2 hours, then treated with the selective FLT3 inhibitor cpd.102
(1 mM) for 1 hour or left untreated (cpd.102 2, washout 2). Then cells were washed twice with medium again containing cpd.102 (cpd. 102 +, washout 2) or
vehicle. At the indicated time points after washing and further incubation, cells were lysed and subjected to a Ras–GTP pulldown. The activation status of Ras, and
ERK was sequentially determined in the same lysates, as described in the Materials and Methods. FLT3 activation was assessed in separate experiments
under identical conditions. The same membrane sections were reprobed to detect the different Ras isoforms and to detect total proteins with pan-specific
antibodies. Antibodies used for immunoprecipitation (IP) or immunoblotting (left side of panels) are indicated. pERK1/2 denotes anti-phospho-p44/42 MAPK;
pTyr591, anti-phospho-FLT3 tyrosine 591. Six (A) or three (B) independent experiments, yielded consistent results. (C) Quantification of Ras-GTP levels in 32D
mFLT3 ITD cells. Five blots for K-Ras and four blots for N-Ras were quantified by densitometry, and intensities of Ras-GTP after inhibitor cpd.102
treatment were normalized to levels without treatment.
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Fig. 5. FLT3 ITD causes constitutive K-Ras
activation at the plasma membrane. 32D cells
expressing human FLT3 ITD, or human MV4-11 cells
endogenously expressing FLT3 ITD (as indicated) were
co-transfected with plasmids encoding either mCherry–
K-Ras or –N-Ras (red) together with the E3-R3(A/D)
reporter for Ras–GTP (green, RBD). Cells were
deprived of serum for 2 hours, treated with the selective
FLT3 inhibitor cpd.102 or left untreated, and subjected
to image analysis by confocal laser scanning
microscopy. (A) Representative images of FLT3-ITD-
expressing 32D cells co-transfected with the mCherry–
K-Ras-encoding plasmid and E3-R3(A/D), and treated
with the selective FLT3 inhibitor cpd.102 or left
untreated, as indicated. (B) Corresponding quantitative
comparisons of cpd.102-treated and non-treated cells.
(C) A 32D cell line harboring an ITD version that is
anchored to the endoplasmic reticulum by a C-terminal
tag (FLT3 ITD R3) was also analyzed.
(D,E) Representative images of (D) MV4-11 cells co-
transfected with mCherry–K-Ras-encoding plasmid and
E3-R3 and (E) corresponding quantitative comparisons
of cpd.102-treated and non-treated cells.
(F,H) Representative images of FLT3-ITD-expressing
32D cells (F) or MV4-11 cells (H) co-transfected with
the mCherry–N-Ras-encoding plasmid and E3-R3
(A/D), and treated with the selective FLT3 inhibitor
cpd.102 or left untreated, as indicated.
(G,I) Corresponding quantitative analysis of the effect
of inhibitor treatment on N-Ras activation in
intracellular compact structures (‘spots’).
(J,K) Quantitative analysis of Ras isoform activation in
different compartments in 32D cells expressing FLT3
ITD (J) or MV4-11 cells (K) transfected with either
mCherry–K-Ras- or mCherry–N-Ras-encoding
plasmids and E3-R3(A/D). PM, plasma membrane. For
all quantifications and statistic analyses, 60 cells from
three independent experiments were analyzed for each
condition. Scale bars: 10 mm. Original magnification
636. Note that some of the probe accumulates in the
nucleus. The reason for this is not known, but it is
unrelated to Ras activation (Augsten et al., 2006).
*P,0.05; **P,0.01; n.s., not significant.
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The presented findings illustrate that K-Ras and N-Ras are
equally well activated by ligand stimulation of wild-type FLT3.Prominent K-Ras and N-Ras activation that could be causallylinked to FLT3 receptor signaling, occurred at the PM. Some
intracellular N-Ras activation may occur at late time-points ofstimulation, possibly reflecting internalization of PM-associatedN-Ras–GTP and its accumulation at an endosomal compartment.Consistent with this notion, partially overlapping localization of
sites of intracellular N-Ras activation with an endosomal markerwas observed in our study. A similar endocytotic process hasbeen proposed to mediate the accumulation of K-Ras at early
endosomes (Lu et al., 2009) and the presence of (active) H-Ras inthe endocytic compartment in the context of growth factorsignaling has also been documented in a number of cases
(Fehrenbacher et al., 2009).
FLT3 ITD was able to activate Ras in the absence of anyligand stimulation. However, the biochemically detected steady-state levels of activated Ras in unstimulated FLT3-ITD-harboring
cells were typically relatively low and only basal K-Ras but notbasal N-Ras seemed to be under control of FLT3 ITD. Our dataon the effects of transient receptor blockade indicate that a
negative feedback mechanism downstream of FLT3 ITD keepsRas–GTP levels at bay in these cells.
Several aspects of the topology of Ras activation by FLT3 ITDdisclosed in the present study warrant special mention. Notably,
despite the dominant intracellular localization of FLT3 ITD [datain Fig. 1 and published findings (Koch et al., 2008; Schmidt-Arraset al., 2009; Schmidt-Arras et al., 2005)], K-Ras activation
proceeded at the PM. Accordingly, complete intracellular retentionof FLT3 ITD using an ER-retention tag (‘R3 anchor’), abolishedK-Ras activation (Fig. 5A,C). We conclude that the PM-bound
pool of FLT3 ITD is necessary and sufficient to activate K-Ras atthis location. Activation of Ras by FLT3 ITD at the PM isconsistent with previous signaling analyses of differently localizedFLT3 ITD pools. For example, FLT3 ITD, which was forced into
an ER compartment by treatment of cells with Brefeldin A or byemploying the aforementioned ER-specific targeting sequence,largely lost the ability to activate Erk1/2 (Choudhary et al., 2009;
Schmidt-Arras et al., 2009).
N-Ras activation at the PM by FLT3 ITD was much lessefficient than activation of K-Ras, at least in the employedsettings using overexpressed Ras proteins. A possibly lower
potency of FLT3 ITD in terms of N-Ras activation at the PMcould be linked to the segregation of both proteins into differentPM microdomains. Differential localization of Ras isoforms in
micro- and nanodomains is extensively documented (Asheryet al., 2006; Omerovic et al., 2008). Of note, some apparentintracellular N-Ras–GTP formation was observed in FLT3-ITD-
expressing cells. Since the appearance of those compactstructures labeled by E3-R3(A/D) was, however, not reversedby FLT3 kinase inhibition, we hypothesize that it was, at least
primarily, an incidental consequence of N-Ras and/or E3-R3(A/D) probe overexpression.
Cell biological experiments were performed to assess the roleof Ras in FLT3-ITD-dependent signaling. The siRNA-based
knockdown of N-/K-Ras revealed an unexpectedly low impact onFLT3-ITD-mediated cell survival and proliferation, probably dueto residual Ras levels. The crucial role of Ras in FLT3-ITD-
driven cell proliferation was, however, obvious in experimentsusing the dominant negative K-RasS17N as an alternative meansof blocking Ras signaling. A profound requirement of Ras for
Fig. 6. Inhibition of FLT3-ITD-driven cell proliferation and induction
of apoptosis by interference with Ras activation at the plasma
membrane. (A) Dn K-RasS17N inhibits FLT3-ITD-dependent cell
proliferation. MV4-11 cells were transduced with lentiviral particles
driving expression of EYFP-tagged wild-type (WT) K-Ras, dn K-
RasS17N, or the yellow fluorescent control protein Venus, as indicated.
The fraction of cells in S phase was determined by DNA labeling using a
Click-iT DNA synthesis kit after 72 hours. Alexa Fluor 647 labeling was
scored for the EYFP/Venus positive cells (n54, means 6 s.e.m.;
*P,0.05, **P,0.01, t-test). (B,C) Apoptosis induction by dn K-
RasS17N. MV4-11 cells transduced with the indicated expression
constructs were subjected to flow cytometric apoptosis assays using the
annexin-V PE method. (B) Example of analysis. (C) Quantification of
multiple experiments. The numbers represent the fraction of yellow
fluorescent cells that were positive for PE-labeled annexin-V (n54, means
6 s.e.m.; *P,0.05, **P,0.01, t-test).
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survival and FLT3-ITD-induced DNA synthesis in these cells
was detected. It is likely that this effect is at least partially
mediated by preventing Erk1/2 activity, which is prominently
dependent on FLT3 ITD signaling (Fig. 4) and downstream Ras
activation (supplementary material Fig. S3). Furthermore, Ras
downstream pathways, such as the phosphoinositide 3-kinase
pathway, may also be involved, but have not been explored in
this study. Since K-RasS17N is localized exclusively at the PM
(Choy et al., 1999) and can only interfere with Ras signaling at
this location, these data further underscored the critical role of
plasma-membrane-bound Ras activation for FLT3 ITD signaling.
In conclusion, this study demonstrates that both wild-type
FLT3 and FLT3 ITD can potently activate Ras in leukemic cells
and that Ras activation is essential for proliferation and survival
of FLT3-ITD-expressing AML cells. Moreover, in the light of
recent data pointing to the existence of distinct signaling
platforms for FLT3 ITD at the PM and endomembranes of
myeloid leukemia cells (Choudhary et al., 2009; Schmidt-Arras
et al., 2009), our present findings reinforce the notion that
activation of Ras and Ras-effector pathways such as the Raf–
MEK–Erk axis in the context of FLT3-ITD-dependent
transformation originates at least predominantly at the PM.
The features of FLT3-ITD-mediated Ras activation described in
the current study may be of broader relevance. Spatial features of
Ras activation and its potential biological importance have not yet
been assessed for any of the intracellular retained RTKs. We
propose that Ras activation downstream of other activated and
intracellular retained RTKs, e.g. activated c-Kit in gastrointestinal
stroma tumors (Tabone-Eglinger et al., 2008) or activated ALK in
neuroblastoma tumors (Mazot et al., 2011) may exhibit similar
features, a hypothesis which can be tested using the methodology
used in the present study.
Materials and MethodsAntibodies and reagents
Anti-pan-Ras (Ab-4) was from Oncogene Science (Cambridge, MA, USA). H-Ras(F235), K-Ras (F234) and N-Ras (F155) antibodies were purchased from Santa CruzBiotechnology (Santa Cruz, CA, USA). For the simultaneous detection of K-Ras andN-Ras, a mixture of anti-pan-Ras (Ab-4) and anti-N-Ras (F155) antibodies was used,which was titrated for detection of all Ras isoforms with similar sensitivity usingrecombinant proteins. This antibody preparation is designated ‘anti-pan-Ras’antibody throughout the manuscript. Polyclonal rabbit anti-FLT3 S-18 (used forimmunoprecipitation of human FLT3) and C-20 (used for immunoblotting of humanFLT3) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).Polyclonal goat anti-FLT3 antibody used for immunoprecipitation andimmunoblotting of murine FLT3 (AF768) and monoclonal anti-human FLT3(MAB812) for immunostainings were obtained from R&D Systems (Wiesbaden,Germany). For flow cytometric analysis, R-phycoerythrin (R-PE)-conjugated mousemonoclonal anti-CD135 (hFLT3; BD, Heidelberg, Germany; catalog no. 558996)was used. Pan-Erk (monoclonal) and ERK1 (#M12320) antibodies were purchasedfrom BD Transduction Laboratories (Heidelberg, Germany). Antibodies recognizingphospho-FLT3 Y591 (#3461), total phosphotyrosine (pY100, #9411), and phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204; #9106), were obtained from Cell Signaling(Frankfurt, Germany), anti-b-actin antibody (A5441) was purchased from Sigma-Aldrich (Taufkirchen, Germany).
Horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG was obtainedfrom KPL (Gaithersburg, MD, USA). Wheat germ agglutinin (WGA) conjugatedto Alexa Fluor 488 was from Life Technologies/Molecular Probes (Darmstadt,Germany). Glutathione S-transferase (GST)–Raf-1 (v-raf-1 murine leukemia viraloncogene homologue 1)–RBD (Ras binding domain) (GST–RBD) was producedby expression in Escherichia coli using standard procedures. Cpd.102, abisindolylmethanone-type inhibitor that is selective for FLT3, was previouslydescribed (Mahboobi et al., 2006). Recombinant human FLT3 ligand (FL) andmurine IL3 were purchased from Peprotech Ltd (London, UK).
Plasmids
PmCherry-K-Ras and pmCherry-N-Ras were generated as previously described(Rubio et al., 2010). A previously characterized multivalent fluorescent reporter of
Ras–GTP, based on the trimerization of the Ras binding domain (RBD) of c-Raf,was used to visualize Ras–GTP in live cells (Augsten et al., 2006; Rubio et al.,2010). Throughout this study we used EGFPx3-RBDx3 (abbreviated: E3-R3) andthe attenuated version EGFPx3-RBD(R59A/N64D)x3 [abbreviated: E3-R3(A/D)],as indicated in the figure legends.
Constructs for lentiviral expression of wild-type human K-Ras and the dn K-RasS17N were obtained by standard cloning procedures. EYFP-K-Ras wasgenerated by replacing mCherry in mCherry–K-Ras (Rubio et al., 2010) withEYFP through AgeI–BsrGI restriction cloning. The S17N mutation was introducedby site-directed mutagenesis using the QuikChangeTM (Agilent, Waldbronn,Germany) method. Thereafter, the sequences encoding the corresponding EYFP–Ras fusion proteins were transferred into the vector LeGO-iG2 (Weber et al.,2008). The cloning strategy led to removal of the IRES-EGFP cassette of LeGO-iG2 (details available on request). As a reference vector LeGO-iV was used,driving expression of the yellow fluorescent protein Venus (http://www.lentigo-vectors.de/vectors.htm).
Cell lines
32D cells stably expressing murine Flt3 wild-type (32D Flt3 wt) or murine Flt3with human ITD (Mizuki et al., 2000) (32D Flt3 ITD) were kindly provided by DrRebekka Grundler and Prof. Justus Duyster (Technical University Munich,Germany). Parental 32D cells, and 32D cells stably expressing human FLT3 wild-type (32D hFLT3 wt), or the human FLT3 ITD variant (32D hFLT3 ITD) werekindly provided by Prof. Hubert Serve (University of Munster, Germany). RS4-11cells and MV4-11 cells were purchased from the Deutsche Sammlung furMikroorganismen und Zellkulturen (DSMZ), Braunschweig, Germany. All cellswere maintained in RPMI 1640 medium supplemented with 10% heat-inactivatedFCS (BioWest, Essen, Germany). The medium for 32D cells was additionallysupplemented with 20 mM HEPES, 1 mM sodium pyruvate and 1 ng/ml mIL3.All cell lines were cultured at 37 C in a 5% CO2 atmosphere.
Immunodetection of FLT3 in leukemia cell lines
Detection of FLT3 localization by immunocytochemistry in RS4-11 and MV4-11cells was done as previously described (Schmidt-Arras et al., 2009). In brief, thePM was stained on ice with WGA conjugated to Alexa Fluor 488 (diluted 1:1000);cells were then fixed with paraformaldehyde, quenched with ammonium chloride,and permeabilized with 0.2% Triton X-100. Unspecific staining was blockedwith 10% FCS in PBS. Cells were incubated for 1 hour at room temperature withanti-FLT3 antibodies (diluted 1:50), washed in PBS, and stained for 1 hour in thedark with Alexa-Fluor-555-conjugated secondary antibodies. Nuclei were stainedwith Hoechst 33342. The slides were then mounted with mounting solution andexamined on a confocal Olympus FV 1000 microscope (Olympus, Hamburg,Germany) with a PlanApo 606/1.4 NA oil objective or a 606/1.35 NA oilobjective, or on a Leica SP5 microscope (Leica Microsystems GmbH, Wetzlar,Germany) with a HCX PLAPO lambda blue 63.06 1.4 oil UV objective. Cellstainings to detect surface localized or total FLT3 by flow cytometry wereperformed exactly as described earlier (Schmidt-Arras et al., 2009). Cells wereanalyzed with a FACS Canto cytometer (Becton Dickinson, Heidelberg, Germany)and results were analyzed using FlowJo software (Tree Star, Ashland, OR, USA).
Ras-GTP pulldown assay, and FLT3 immunoprecipitation
Cells were grown to a density of 106 cells/ml and were deprived of serum (for thetime indicated in the figure legends) in RPMI 1640 supplemented with 0.2% fatty-acid-free/endotoxin-low BSA, 1 mM sodium pyruvate and 50 mM HEPES pH 7.5,counted, and then resuspended at 26107 cells/ml in the same solution. Cellsuspensions were incubated in a water bath at 37 C and tubes were flipped every2–3 minutes. After appropriate stimulation of wild-type FLT3-expressing cellswith FL (20 ng/ml) or at the appropriate time after washing out cpd.102 fromFLT3 ITD cells (see figure legends for details), 1 ml of the cell suspension wastransferred to a 1.5 ml reaction vial, and quickly spun in a table-top centrifuge. Themedium was aspirated, and the cell pellet was lysed with 1 ml ice-cold lysis buffer(50 mM HEPES pH 7.5, 140 mM NaCl, 5 mM MgCl2, 1 mM DTT, 1% NP-40)supplemented with protease and phosphatase inhibitors, 100 mM GDP and 30 mgGST–RBD protein. GDP and GST–RBD were included at this point to quenchpost-lytic GTP loading or GAP-dependent Ras-bound GTP hydrolysis,respectively. Cell extracts were cleared by centrifugation at 14,000 rpm at 4 Cfor 20 minutes, and GST–RBD/Ras–GTP complexes were collected on 40 mlglutathione–Sepharose beads (1:1 suspension) by incubation with end-over-endrotation at 4 C for 30 minutes. Beads were washed once with 500 ml lysis buffer,supplemented with protease and phosphatase inhibitors. Bound proteins wereeluted with SDS-PAGE sample buffer and processed for immunoblotting. Thesupernatant of the Ras–GTP pulldown, once GST–RBD had been collected onglutathione–Sepharose was used to obtain total cell-lysate aliquots forimmunoblotting. FLT3 immunoprecipitations were either performed using thesame lysates or lysates prepared independently under identical conditions with0.5 mg anti-mFLT3 antibody or 0.8 mg anti-hFLT3 antibody, and protein-G– or -A–Sepharose beads, respectively.
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ImmunoblottingProtein samples were separated on 12.5% (cell lysates, Ras–GTP pulldown) or 5%(FLT3 immunoprecipitation) SDS-PAGE gels, and transferred to HybondTM-CExtra nitrocellulose membranes (GE Healthcare Life Sciences, Freiburg,Germany). The membranes were incubated with primary antibody at 4 Covernight, followed by development with horseradish peroxidase (HRP)-conjugated secondary antibodies and enhanced chemiluminescence detection.Images were acquired with a LAS 4000 CCD camera system (Fujifilm, Dusseldorf,Germany), quantified with Multi Gauge V3.0 software (Fujifilm) and processedusing Adobe Photoshop CS3 extended Version 10.0 (Adobe Systems, San Jose,CA).
Plasmid transfection and confocal live-cell imaging
For transfection, 66106 cells were washed once in RPMI 1640 medium containing10% FCS, then mixed with 10 mg of plasmid DNA in 300 ml culture medium in0.4-cm gap cuvettes and electroporated using a GenePulserII instrument (BioRad,Hercules, USA), with settings of 300 V and 960 mF and resistance set to infinity.24 hours after transfection, living cells were examined with a Zeiss LSM 510Axiovert confocal microscope (Carl ZEISS GmbH, Jena, Germany) equipped witha thermostated stage chamber (IBIDI, Munchen, Germany) at 37 C. Confocalimages (1 mm optical slice) were acquired using a 636water immersion objectivelens. mCherry and EGFP were excited with the HeNe 543 nm and the argon488 nm line, respectively, in subsequent tracks. Emitted fluorescence wascollected with a 560 nm longpass and a 505–550 nm bandpass filter, respectively.
Prior to experiments, cells were deprived of serum in RPMI 1640 supplementedwith 50 mM HEPES (pH 7.4), 1 mM sodium pyruvate, and 0.2% endotoxin-low,fatty acid-free BSA (Sigma-Aldrich, Taufkirchen, Germany) for 2 hours.
FLT3-ITD-expressing MV4-11 cells or 32D cells were treated with 1 mMcpd.102 for the last 45 minutes of the starvation period, or left untreated. Cellswere plated on poly-L-lysine-coated (poly-L-Lysine HBr, MV 30,000–70,000;Sigma) glass-bottomed (coverslip) 35-mm dishes 5 minutes before monitoring.Confocal images of at least 20 cells per experiment were recorded. Only cells withclear mCherry–Ras signals at the PM were considered for the analysis.
Wild-type FLT3-expressing RS4-11 cells were placed under the microscope,then challenged with FL (100 ng/ml), and then a timed series of confocal imagesof selected cells were acquired.
All images of a time series (for RS4-11 cells) or of experimental groups of cellsfor comparison (for MV4-11 and 32D hFLT ITD cells) were exported as LSMfiles, subjected to the same processing routine using Zeiss ZEN 2008 Light Editionsoftware (ZEISS, Jena, Germany), converted to TIFF files and processed againidentically using Adobe Photoshop CS3 extended Version 10.0 (Adobe Systems,San Jose, CA). For presentation in the figures, parts of the images were enlarged toreveal more details. Scale bars are depicted accordingly.
Role of Ras signaling for cell proliferation and viabilitySiRNAs were ON-TARGETplus SMART pools (Thermo Scientific, Dharmacon,Schwerte, Germany): control pool/non-targeting pool, cat. no. D-001810-10-20;human KRAS (KRAS), cat. no. L-005069-00-0005; human NRAS (NRAS), cat. no.L-003919-00-0005; human FLT3 (hFLT3), cat. no. L-003137-00-0005).Transfection was performed using the nucleofection device (Amaxa Inc.,Cologne, Germany) with NucleofectorH kit V and the program A-30, accordingto the manufacturer’s instructions. After 72 hours of culture, proliferation/viabilityassays were performed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) as described previously (Choudhary et al., 2009). The values werenormalized to the control, mock-transfected cells. Cell cycle analysis, using FITC–BrdU staining, was performed 48 hours after siRNA transfection or inhibitortreatment, as indicated. For BrdU incorporation a FITC BrdU Flow Kit (BDPharmingen, Heidelberg, Germany) was used according to the manufacturer’sinstructions.
Preparation of lentiviral particles and transduction of MV4-11 cells withconstructs encoding EYFP–K-Ras or EYFP–K-RasS17N in the vector LeGO-iG2or control transductions with LeGO-iV were performed as described previously(Arora et al., 2011). Transduced cell pools were analyzed for cell proliferationusing a Click-iT DNA synthesis kit (Alexa-Fluor-647-labeled C10419, LifeTechnologies, Darmstadt, Germany), or for apoptosis using an Annexin V-PE Kit(559763, BD Pharmingen, Heidelberg, Germany) with a FACSCanto flowcytometer (BD Biosciences, Heidelberg, Germany). Transduced cells wereselected for analysis based on EYFP/Venus expression.
Statistical analyses
The relative frequencies of FLT3-ITD-expressing cells with certain features of Rasactivation (overlapping localization of mCherry–Ras and E3-R3/E3-R3(A/D)signals) were ascertained. Frequencies from three experiments were summed, andthe differences of different conditions were checked by use of a x2-test. Theseanalyses yielded cumulative integer values of cells in the respective groups andtherefore no error bars are shown. Corresponding calculations were done withSPSS version 17.0 software (IBM, Somers, NY, USA). Data for biological assays(proliferation, cell cycle analysis, apoptosis) are expressed as means 6 s.e.m. For
comparison of groups in these assays, the two-tailed t-test was used. Calculationswere done with SigmaPlot 11.0 software (Systat Software Inc., Chicago, IL, USA).P,0.05 (*) or P,0.01 (**), as indicated in the figure legends, was regarded asstatistically significant.
AcknowledgementsWe thank Shu-Ping Song, Ute Wittig and Katja Schubert fortechnical help, Christoph Biskup for help with some imageacquisitions, and Christoph Kaether for provision of a reagent.
Author contributionsS. Kothe, I.R., and F.D.B. conceived and designed the experimentsand wrote the manuscript. S. Kothe, J.P.M., S.A.B, T.T., M.F., S.Koch., C.T., R.P.R. and I.R. performed experiments and analyzeddata. F.D.B. directed the project.
FundingThis work was supported by the Deutsche Forschungsgemeinschaft[grant numbers BO 1043/7-1 to F.D.B., RU 860/3-1 to I.R.]; and theDeutsche Krebshilfe [collaborative grant number 108401 TP2 toF.D.B.].
Supplementary material available online at
http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.131789/-/DC1
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Ras activation by FLT3 ITD 4755